A novel auxin conjugate hydrolase from wheat with substrate specificity for longer side-chain auxin amide conjugates

Abstract

This study investigates how the ILR1-like indole acetic acid (IAA) amidohydrolase family of genes has functionally evolved in the monocotyledonous species wheat (Triticum aestivum). An ortholog for the Arabidopsis IAR3 auxin amidohydrolase gene has been isolated from wheat (TaIAR3). The TaIAR3 protein hydrolyzes negligible levels of IAA-Ala and no other IAA amino acid conjugates tested, unlike its ortholog IAR3. Instead, TaIAR3 has low specificity for the ester conjugates IAA-Glc and IAA-myoinositol and high specificity for the conjugates of indole-3-butyric acid (IBA-Ala and IBA-Gly) and indole-3-propionic-acid (IPA-Ala) so far tested. TaIAR3 did not convert the methyl esters of the IBA conjugates with Ala and Gly. IBA and IBA conjugates were detected in wheat seedlings by gas chromatography-mass spectrometry, where the conjugate of IBA with Ala may serve as a natural substrate for this enzyme. Endogenous IPA and IPA conjugates were not detected in the seedlings. Additionally, crude protein extracts of wheat seedlings possess auxin amidohydrolase activity. Temporal expression studies of TaIAR3 indicate that the transcript is initially expressed at day 1 after germination. Expression decreases through days 2, 5, 10, 15, and 20. Spatial expression studies found similar levels of expression throughout all wheat tissues examined.

Figure 1.

Neighbor-joining phylogram of Arabidopsis ILR1-like protein family members orthologous to TaIAR3. The wheat hydrolase (bold) demonstrates the closest homology to the IAR3 protein sequence (bold); the high bootstrap value at the node of the tree (bold) indicates a probability score of approximately 98% for the validity of this prediction. The scale at the bottom of the figure indicates relative genetic distance.

Figure 2.

HPLC separation of the substrate, IBA-Ala, and the reaction product, IBA, after incubation of the substrate with E. coli cell lysate for 1 h. Chromatograms were recorded at 280 nm. A, Standards of IBA and IBA-Ala. B, Lysate of cells containing an empty vector. C, Lysate of cells containing the peTaIAR3 vector uninduced. D, Lysate of cells containing the peTaIAR3 vector induced with IPTG.

Figure 3.

Free and total IAA and IBA concentrations in wheat during seedling development. The values presented are means ± se of three independent experiments.

Figure 4.

Temporal (A) and spatial (B) expression of TaIAR3 transcripts. Graphs were generated using the relative standard curve method (Applied Biosystems, 2001). A, Calibrator was the 1-d-old plant expression data. B, Calibrator was the coleoptile expression data.